Measurement of Oxidative Damage at Pyrimidine Bases in γ-Irradiated DNA

Oxidized nucleobases represent one of the main classes of damage induced in DNA by ionizing radiation. Emphasis was placed in this work on the measurement of four oxidized pyrimidine bases, including 5-(hydroxymethyl)uracil (5-HMUra), 5-formyluracil (5-ForUra), 5-hydroxycytosine (5-OHCyt), and 5-hydroxyuracil (5-OHUra), in isolated DNA upon exposure to gamma radiation in aerated aqueous solution. For this purpose, both high performance liquid chromatography associated with electrochemical detection (HPLC-EC) and gas chromatography coupled to mass spectrometry (GC-MS) were used. Conditions of hydrolysis of the N-glycosidic bond were carefully checked in order to achieve a quantitative release of the lesions. We showed that 60% formic acid treatment leads to the decomposition of the four lesions studied. On the other hand, hydrolysis based on the use of either 88% formic acid or 70% hydrogen fluoride in pyridine (HF/Pyr) allowed the quantitative release of the modified bases, with the exception of 5-HMUra when the latter reagent was utilized. A dose course study of the radiation-induced formation of 5-HMUra and 5-ForUra in DNA by using the GC-MS assay showed that the latter lesion was produced in a 2.1-fold higher yield than the former one. HF/Pyr and 88% formic acid hydrolysis provided similar results for 5-ForUra, indicating the reliability of both techniques for the measurement of this lesion. For 5-OHUra and 5-OHCyt, the level of modification determined by GC-MS analysis was higher after 88% formic acid treatment than upon HF/Pyr hydrolysis. When DNA was enzymatically digested and analyzed by HPLC-EC for 5-OHdCyd and 5-OHdUrd, the results were very close to those obtained by GC-MS following HF/Pyr treatment. It was concluded that additional amounts of both 5-OHUra and 5-OHCyt are produced during the 88% formic acid treatment from radiation-induced 5,6-saturated pyrimidine precursors. It is likely that cytosine and uracil diols are involved in this reaction. The radiochemical yields of formation (in mumol.J-1) for the products studied are in the following decreasing order: 5-ForUra (0.0083) > 5-OHCyt (0.0046) > 5-HMUra (0.0039) > 5-OHUra (0.0035).